Cotton provides much of the high quality fiber for the textile industry. The modification of cotton fiber characteristics to better suit the requirements of the industry is a major effort in breeding by either classical methods or by genetically altering the genome of cotton plants.
About 90% of cotton grown worldwide is Gossypium hirsutum L., whereas Gossypium barbadense accounts for about 8%. As in most flowering plants, cotton genomes are thought to have incurred one or more polyploidization events and to have evolved by the joining of divergent genomes in a common nucleus. The cotton commerce is dominated by improved forms of two “AD” allotetraploid species, Gossypium hirsutum L. and Gossypium barbadense L (both 2n=4x=52). Allotetraploid cottons are thought to have formed about 1-2 million years ago, in the New World, by hybridization between a maternal Old World “A” genome taxon resembling Gossypium herbaceum (2n=2x=26) and paternal New World “D” genome taxon resembling Gossypium raimondii or Gossypium gossypioides (both 2n=2x=26). Wild A genome diploid and AD allotetraploid Gossypium taxa produce spinnable fibers. One A genome diploid species, Gossypium arboreum (2n=2x=26), remains intensively bred and cultivated in Asia. Its close relative and possible Gossypium progenitor, the A genome diploid species G. herbaceum, also produces spinnable fiber. Although the seeds of D genome diploids are pubescent, none produce spinnable fibers. No taxa from the other recognized diploid Gossypium genomes (B, C, E, F, G and K) have been domesticated. Intense directional selection by humans has consistently produced AD allotetraploid cottons that have superior yield and/or quality characteristics compared to the A genome diploid cultivars. Selective breeding of G. hirsutum (AADD; “Upland” cotton) has emphasized maximum yield, whereas G. barbadense (AADD; “Sea Island”, “Pima”, or “Egyptian” cotton) is prized for its fibers of superior length, strength, and fineness (Jiang et al., 1998, Proc Natl Acad Sci USA. 95(8): 4419-4424).
A cotton fiber is a single cell that initiates from the epidermis of the outer integument of the ovules, at or just prior to anthesis. Thereafter, the fibers elongate rapidly for about 3 weeks before they switch to intensive secondary cell wall cellulose synthesis. Fiber cells interconnect only to the underlying seed coat at their basal ends and influx of solute, water and other molecules occurs through either plasmodesmata or plasma membrane. Ruan et al. 2001 (Plant Cell 13: 47-63) demonstrated a transient closure of plasmodesmata during fiber elongation. Ruan et al. 2004 (Plant Physiology 136: 4104-4113) compared the duration of plasmodesmata closure among different cotton genotypes differing in fiber length and found a positive correlation between the duration of the plasmodesmata closure and fiber length. Furthermore, microscopic evidence was presented showing callose deposition and degradation at the fiber base, correlating with the timing of plasmodesmata closure and reopening. Expression of a endo-1,3-beta-glucanase gene in the fibers, allowing to degrade callose, correlated with the reopening of the plasmodesmata at the fiber base.
WO2005/017157 describes methods and means for modulating fiber length in fiber producing plants such as cotton by altering the fiber elongation phase. The fiber elongation phase may be increased or decreased by interfering with callose deposition in plasmodesmata at the base of the fiber cells.
WO2008/083969 (claiming priority of European patent application EP 07000550) discloses isolated DNA molecules comprising a nucleotide sequence encoding cotton endo-1,3-beta-glucanases and fiber cell preferential promoter or promoter regions, as well as methods for modifying the length of a fiber of a cotton plant using these sequences or promoters. WO2008/083969 also describes that the timing of expression of the A and D subgenome specific alleles of the fiber specific endo-1,3-beta-glucanase gene in Gossypium hirsutum is different. Whereas the onset of the expression of the D subgenome specific allele correlates with the end of the rapid elongation phase (about 14 to 17 days post-anthesis, hereinafter abbreviated “DPA”), onset of the expression of the A subgenome specific allele is delayed until the beginning of the late fiber maturation phase (about 35-40 DPA) depending on growth conditions.
One fiber characteristic that is of special interest for the cotton industry is fiber strength. There is not only a high correlation between fiber strength and yarn strength, but also cotton with high fiber strength is more likely to withstand breakage during the manufacturing process.
Fiber strength is, among many other textile properties of cotton fibers (e.g., fiber wall thickness or maturity, dyeability, extensibility . . . ), described to be directly dependent on the amount and properties (e.g., degree of polymerization, crystallite size, and microfibril orientation) of cellulose (Ramey, 1986, In: Mauney J. R. and Stewart J. McD. (eds.) Cotton Physiology. The Cotton Foundation, Memphis, Tenn., pp. 351-360; Triplett, 1993, In: Cellulosics: Pulp, Fibre, and Environmental Aspects. Ellis Horwood, Chichester, UK, pp. 135-140; Hsieh, 1999, In: Basra A. S. (ed.) Cotton Fibers: Developmental Biology, Quality Improvement, and Textile Processing. The Haworth Press, New York, pp. 137-166). Advances in the past decade, particularly using the model plant Arabidopsis (Arioli et al., 1998, Science 279(5351): 717-720), have led to a great increase in the knowledge of the proteins involved in cellulose synthesis. Despite this, there is still much to learn about cellulose synthesis, especially about how it is regulated at both transcriptional and post-transcriptional levels (Taylor, 2008, New Phytologist 178 (2), 239-252).
Typical primary fiber cell walls in G. hirsutum, which are about 0.5 μM thick and contain 20-25% cellulose along with pectin, xyloglucan, and protein (Meinert and Delmer 1977, Plant Physiol 59:1088-1097), are synthesized during fiber elongation (Haigler, 2007, In: R. M. Brown, Jr. and I. M. Saxena (eds.), Cellulose: Molecular and Structural Biology, 147-168, Springer). Primary wall deposition proceeds alone until 14-17 DPA, then a transition phase with concurrent primary and secondary wall deposition occurs between 15-24 DPA (representing deposition of the “winding layer”), followed by predominantly secondary wall synthesis until at least 40 DPA. The first period of wall thickening (12-16 DPA) is accomplished by continued synthesis in the same proportions of primary wall components (Meinert and Delmer, 1977, supra), an observation that is consistent with increasing wall birefringence while the cellulose microfibrils remain transversely oriented (Seagull, 1986, Can J Bot 64:1373-1381). The secondary wall finally attains a thickness of 3-6 μM around the whole circumference of the fiber, becoming thinner only at the fiber tip. In G. barbadense, there is an overlap between primary and secondary wall deposition within each fiber rather than in the fiber population because the overlapping period is greatly prolonged, and 90% of secondary wall deposition is complete before elongation ceases (DeLanghe, 1986, In: Mauney J. R. and Stewart J. McD. (eds.) cotton Physiology. The Cotton Foundation, Memphis, Tenn., pp. 325-350). It is thought that elongation continues exclusively at the fiber tip as secondary wall is deposited over most of the cell surface.
Maltby et al. (1979, Plant Physiol. 63, 1158-1164) describe that developing fibers of Gossypium hirsutum transiently synthesize 1,3-beta-D-glucan (callose) at the onset of secondary wall deposition followed by massive synthesis of cellulose. Meier et al. (1981, Nature 289: 821-822) describe that callose may be a probable intermediate in biosynthesis of cellulose of cotton fibers. DeLanghe (1986, supra) describes that callose may be required in cotton fiber secondary walls to provide a space for the crystallization and final orientation of cellulose microfibrils in the exoplasmic zone in the absence of typical matrix molecules.
The inventions described hereinafter in the different embodiments, examples, figures and claims provide improved methods and means for modulating fiber strength. More specifically, the present invention describes how to increase fiber strength and at the same time maintain a high fiber yield in plants. In particular, the invention describes how to increase fiber strength in cotton species selected for high yield, such as Gossypium hirsutum, by introgression of fiber strength determining genes from other cotton species selected for high fiber strength, such as Gossypium barbadense. Methods are also provided to identify molecular markers associated with fiber strength in a population of cotton varieties and related progenitor plants. The inventions described hereinafter also provide novel nucleic acid molecules encoding fiber-specific Gossypium glucanase proteins (GLUC1.1) and the proteins as such.